Xylose is the second most abundant sugar in nature (Saha B C: Hemicellulose bioconversion. J Ind Microbiol Biotechnol 2003, 30:279-291), however yeast strains such as Saccharomyces are unable to metabolize xylose. A technical challenge to enable and enhance yeast capability in utilization of pentose sugars such as xylose and arabinose harbored in biomass is to engineer yeast stains that can metabolize those sugars.
Genetic engineering efforts have been made to improve xylose utilization by overexpressing genes encoding pentose phosphate pathway (PPP) enzymes to enhance xylose flux into central carbon metabolism. For native S. cerevisiae, there are no xylose-specific transporters available and xylose uptake is via certain hexose transporters such as Hxt4, Hxt5, Hxt7, and Gal2. Recently, several heterologous sugar transporter genes possessing xylose transport functions have been expressed in recombinant S. cerevisiae such as SUT1, XUT1 or XUT3 from S. stipitis, At5g59250 and At5g17010 from A. thaliana, An25 from N. crassa, DEHA0D02167 and XylHP from D. hansenii, and symporters GXS1 and GXF1 genes from C. intermedia. Improvement of xylose utilization by such efforts was observed but a satisfactory level has not been reached.
While there has been focus on engineering yeast strains that metabolize xylose as a sugar source, the environment yeast operate have both xylose and glucose as part of the batch. For S. cerevisiae strains, uncontrolled, high-level expression of many genes required for xylose fermentation can be detrimental to cell growth and fermentation (Id.) Additionally, certain genes required for efficient xylose fermentation negatively affect glucose fermentation (Meinander N Q, et al., Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1. Bioresource Technol 1999, 68:79-87).
Recombinant production of any heterologous protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein of interest is placed under the control of a promoter suitable for the host cell. The expression cassette is then introduced into the host cell (i.e., usually by plasmid-mediated transformation or targeted integration into the host genome) and production of the heterologous protein is achieved by culturing the transformed host cell under conditions necessary for the proper function of the promoter contained within the expression cassette. Thus, the development of new host cells (e.g., transformed yeast) for recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of a protein of interest in the host cell.
While there are promoters that have been isolated from yeast cells that are useful in heterologous gene expression in yeast, most of these promoters provide constitutive gene expression. There are fewer inducible promoters available from S. cerevisiae for regulating gene expression and none of these are regulated by xylose. Thus, there is a need to develop a promoter that controls gene expression in response to xylose availability. Such an induced promoter would ensure efficient metabolism energy for the yeast cell without the cell growth and fermentation disadvantages stemming from unregulated, high-level expression, of multiple genes.